RSK3 switches cell fate: from stress-induced senescence to malignant progression

Background TGFβ induces several cell phenotypes including senescence, a stable cell cycle arrest accompanied by a secretory program, and epithelial-mesenchymal transition (EMT) in normal epithelial cells. During carcinogenesis cells lose the ability to undergo senescence in response to TGFβ but they maintain an EMT, which can contribute to tumor progression. Our aim was to identify mechanisms promoting TGFβ-induced senescence escape. Methods In vitro experiments were performed with primary human mammary epithelial cells (HMEC) immortalized by hTert. For kinase library screen and modulation of gene expression retroviral transduction was used. To characterize gene expression, RNA microarray with GSEA analysis and RT-qPCR were used. For protein level and localization, Western blot and immunofluorescence were performed. For senescence characterization crystal violet assay, Senescence Associated-β-Galactosidase activity, EdU staining were conducted. To determine RSK3 partners FLAG-baited immunoprecipitation and mass spectrometry-based proteomic analyses were performed. Proteosome activity and proteasome enrichment assays were performed. To validate the role of RSK3 in human breast cancer, analysis of METABRIC database was performed. Murine intraductal xenografts using MCF10DCIS.com cells were carried out, with histological and immunofluorescence analysis of mouse tissue sections. Results A screen with active kinases in HMECs upon TGFβ treatment identified that the serine threonine kinase RSK3, or RPS6KA2, a kinase mainly known to regulate cancer cell death including in breast cancer, reverted TGFβ-induced senescence. Interestingly, RSK3 expression decreased in response to TGFβ in a SMAD3-dependent manner, and its constitutive expression rescued SMAD3-induced senescence, indicating that a decrease in RSK3 itself contributes to TGFβ-induced senescence. Using transcriptomic analyses and affinity purification coupled to mass spectrometry-based proteomics, we unveiled that RSK3 regulates senescence by inhibiting the NF-κΒ pathway through the decrease in proteasome-mediated IκBα degradation. Strikingly, senescent TGFβ-treated HMECs display features of epithelial to mesenchymal transition (EMT) and during RSK3-induced senescence escaped HMECs conserve EMT features. Importantly, RSK3 expression is correlated with EMT and invasion, and inversely correlated with senescence and NF-κΒ in human claudin-low breast tumors and its expression enhances the formation of breast invasive tumors in the mouse mammary gland. Conclusions We conclude that RSK3 switches cell fate from senescence to malignancy in response to TGFβ signaling. Supplementary Information The online version contains supplementary material available at 10.1186/s13046-023-02909-5.

were infected with pWZL/RSK3 (RSK3) or control vector pWZL (Ctrl), selected for 1 week with neomycin and plated for indicated assays, followed by TGFβ treatment at 0.5 ng/mL on the next day.(A) EdU pulse.Cells were plated at 1.5 × 10 3 cells/well in 96-well plates, 2 days after treatment with TGFβ EdU was added for 2 h before EdU staining (n = 3 independent experiments).(B to C) RT-QPCR was performed 48 h after TGFβ treatment (n = 4 independent experiments).Ratio paired ttest was used to determine statistical significance.

Figure S1 .
Figure S1.RSK3 inhibits TGFβ-induced proliferation arrest.HMECT were infected with pWZL/RSK3 (RSK3) or control vector pWZL (Ctrl), selected for 1 week with neomycin and plated for indicated assays, followed by TGFβ treatment at 0.5 ng/mL on the next day.(A) EdU pulse.Cells were plated at 1.5 × 10 3 cells/well in 96-well plates, 2 days after treatment with TGFβ EdU was added for 2 h before EdU staining (n = 3 independent experiments).(B to C) RT-QPCR was performed 48 h after TGFβ treatment (n = 4 independent experiments).Ratio paired ttest was used to determine statistical significance.

Figure S4 .Figure S5 .Figure S6 .Figure S7 .
Figure S4.(A) MS-based proteomic characterization of RSK3 interactome.Volcano plot displaying the differential abundance of proteins in FLAG-RSK3 and FLAG co-IP eluates analyzed by MS-based label-free quantitative proteomics.The volcano plot represents the -log10 (limma p-value) on y axis plotted against the log2(Fold Change FLAG-RSK3/FLAG) on x axis for each quantified protein.Green dots represent proteins found significantly enriched in FLAG-RSK3 eluates (Fold change ≥ 5 and p-value ≤ 0.01, leading to a Benjamini-Hochberg FDR < 1 %).(B) Western blot characterization of pIκBα and IκBα levels.Cells were treated with 40µM MG132 2 h prior stimulation with TNFα for 5 min.

Table S1 .
List of primers used for RT-QPCR.

Table S2 .
List of antibodies used.

Table S4 .
Results of TFacts analysis.Common genes: up-regulated in HMECT-pWZL treated with TGFβ compared to non-treated control (TGFβ UP) and downregulated in HMECT-pWZL/RSK3 treated with TGFβ compared to HMECT-pWZL treated with TGFβ (RSK3 DOWN), or the opposite were used for identification of transcription factors.

Table S5 .
Pathway analysis of RSK3 binding proteins.Analysis was performed with DAVID online software.

Table S7 .
Analysis of the METABRIC breast cancer database.Correlation coefficients between expression of indicated epithelial or mesenchymal factors and RSK3.p-values were determined by linear regression analysis, green means negative correlation and red positive correlation.